Qinyuan Wu

New mass spectrometric methods for the study of noncovalent associations of biopolymers

The use of electrospray ionization–mass spectrometry(ESI–MS) for the characterization of noncovalent complexes of biomacromolecules in solution is based upon the gentle nature of the electrospray process that allows a wide range of associations to be transferred intact to the gas phase as fully desolvated complexes. Examples include multimeric proteins, oligonucleotide duplexes, DNA–drug complexes and enzyme-inhibitor complexes. Various studies have indicated that at least some qualities of the three-dimensional solution structures are retained in the gas phase. Recent investigations have also shown the relative stabilities of complexes in the gas phase can be very different than the same complexes in solution. In spite of this, the use of very gentle electrospray interface conditions can provide a direct reflection of relative solution abundances for similar complexes. Competitive binding experiments using sets of ligands have been shown to yield insights regarding relative binding affinities in solution. The potential for high throughput affinity screening of combinatorial libraries using ESI–MS is described based upon the multi-stage MS capability of Fourier transform ion cyclotron resonance instrumentation and involving the characterization of components (after dissociation) of the library constituents initially present as complexes with a target biopolymer in the ion trap. This approach combines, in one rapid experiment, both affinity selection by complex formation with a biopolymer and the identification of the ligands selected from combinatorial mixtures, thus providing information on the relative binding affinities of the library constituents. The present status, limitations and promise of these methods are discussed.

Probing the Energetics of Dissociation of Carbonic Anhydrase-Ligand Complexes in the Gas Phase

This paper describes the use of electrospray ionization-Fourier transform ion cyclotron mass spectrometry (ESI-FTICR-MS) to study the relative stabilities of noncovalent complexes of carbonic anhydrase II (CAII, EC 4.2.1.1) and benzenesulfonamide inhibitors in the gas phase. Sustained off-resonance irradiation collision-induced dissociation (SORI-CID) was used to determine the energetics of dissociation of these CAII-sulfonamide complexes in the gas phase. When two molecules of a benzenesulfonamide (1) were bound simultaneously to one molecule of CAII, one of them was found to exhibit significantly weaker binding (DeltaE50 = 0.4 V, where E50 is defined as the amplitude of sustained off-resonance irradiation when 50% of the protein-ligand complexes are dissociated). In solution, the benzenesulfonamide group coordinates as an anion to a Zn(II) ion bound at the active site of the enzyme. The gas phase stability of the complex with the weakly bound inhibitor was the same as that of the inhibitor complexed with apoCAII (i.e., CAII with the Zn(II) ion removed from the binding site). These results indicate that specific interactions between the sulfonamide group on the inhibitor and the Zn(II) ion on CAII were preserved in the gas phase. Experiments also showed a higher gas phase stability for the complex of para-NO2-benzenesulfonamide-CAII than that for ortho-NO2-benzenesulfonamide-CAII complex. This result further suggests that steric interactions of the inhibitors with the binding pocket of CAII parallel those in solution. Overall, these results are consistent with the hypothesis that CAII retains, at least partially, the structure of its binding pocket in the gas phase on the time scale (seconds to minutes) of the ESI-FTICR measurements.

Specific metal-oligonucleotide binding studied by high resolution tandem mass spectrometry

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) was used to study the binding of metal ions to two oligonucleotides, d(pGCTTGCATp) and d(TTGGCCCTCCTT). Collision-induced dissociation (CID) of the metal-oligonucleotide complex revealed that metal ions preferentially bound to the central thymine region of the sequence. The most probable binding sites were the phosphodiester backbone since the sum of the maximum number of charge addition from the metal ions and the charge state of the whole complex was found to be equal to the number of ionizable protons on the DNA backbone. Although site-specific and sequence-specific binding was observed for all three of the metal ions studied, the binding specificity of UO2(2+) ions was significantly greater than for Mg2+ and Na+. These experiments demonstrate that ESI-MS/MS can be applied to study the binding of metal ions and their complexes to oligonucleotides, providing not only information on the number of metal ions binding to the oligonucleotide, but also information related to the binding site(s) and binding specificity.

On-line Microdialysis Desalting for Electrospray Ionization-Mass Spectrometry of Proteins and Peptides

A novel method has been developed for the rapid desalting of proteins and peptides for electrospray ionization mass spectrometry (ESI-MS). Protein and peptide solutions having high salt concentrations were infused into a microdialysis tube which was directly interfaced with a micro-ESI source. The microdialysis tube was surrounded by a counter-current flow of a buffer solution consisting of 10 mM ammonium acetate. Desalting was completed in one to four minutes for samples containing up to 250 mM NaCl, which would otherwise have required several hours using alternative off-line desalting schemes. The on-line desalting method also increased the signal-to-noise ratio by a factor of more than 40 compared with that obtained without desalting. Detection of subpicomole samples of apomyoglobin having a high salt concentration was also achieved. Initial results for apomyoglobin, albumin and angiotensin I indicate broad applicability of the method.

Application of sequential paired covariance to liquid chromatography-mass spectrometry data enhancements in both the signal-to-noise ratio and the resolution of analyte peaks in the chromatogram

Abstract The algorithm of sequential paired covariance (SPC) has been previously reported to dramatically enhance the signal-to-noise (S/N) ratio for on-line separations combined with mass spectrometry. That initial study focused on a limited number of data sets derived from the combination of capillary electrophoresis (CE) with time-of-flight mass spectrometry using an electrospray interface. Results from the initial study clearly demonstrated that a significant enhancement (almost two orders of magnitude) in the S/N ratio of the eluting peaks in the electropherogram could be obtained, facilitating identification of the analytes. In this report, the algorithm has been applied to liquid chromatography-mass spectrometry data obtained on a triple quadrupole instrument and we have evaluated the general applicability of the SPC approach to several types of microcolumn separations with mass spectrometric detection, including CE coupled with Fourier transform ion cyclotron resonance mass spectrometry. In all the cases we tested, we found the algorithm enhanced the S/N ratios of the resulting chromatograms or electropherograms to a similar extent. This report further demonstrates the SPC approach to enhance the resolution as well as the S/N ratio of the eluting peaks of a complex peptide mixture. While many variations of the algorithm are possible, we have also found higher order covariance (e.g., 3rd order) is useful for eliminating coincidental noise in sequential mass spectra, giving the potential to extract broad, low intensity analyte peaks. We also demonstrate the sequential covariance approach for enhancing the S/N ratio of mass spectra.

The Role of Fourier Transform Ion Cyclotron Resonance Mass Spectrometry in Biological Research — New Developments and Applications

The symbiotic relationship between advances in ionization methods and the improved performance of mass spectrometric instrumentation and its computer control has been key to the growth of mass spectrometry. The new capabilities for the ionization of large molecules introduced in 1988 have, in effect, resulted in a situation where the demands for improved sensitivity, resolution, effective tandem MS capabilities for structural studies, and the online combination with separation methods, are greater than ever and effectively open-ended. This situation coincides with a period in which mass spectrometric hardware has begun a transition from a dominance by quadrupole and sector mass analyzer technologies to other technologies that previously were rarely used for “real world” bioanalytical applications (i. e., time-of-flight mass spectrometers and ion trapping methods).

A HIGH PERFORMANCE LOW MAGNETIC FIELD INTERNAL ELECTROSPRAY IONIZATION-FOURIER TRANSFORM ION CYCLOTRON RESONANCE MASS SPECTROMETER

A new in-magnetic field electrospray ionization (ESI) and Fourier transform ion cyclotron resonance mass spectrometer has been constructed and evaluated. This system is characterized by the use of multiple concentric cryopanels to achieve ultrahigh vacuum in the ion cyclotron resonance cell region, a probe-mounted internal ESI source, and a novel in-field shutter. Initial experiments demonstrate high resolution mass measurement capability at a field strength of 1 T. Mass resolution of 700,000 has been obtained for the 3+ charge state of Met-Lys-bradykinin (at m/z 440) generated by electrospray ionization. When electron impact ionization was employed, resolution in excess of 9,200,000 was achieved for nitrogen molecular ions (N2+). Isotopic resolution for molecular ions of bovine ubiquitin (MW=8565 µ) also was achieved by using small ion populations.

Enhanced accumulated trapping efficiency using an auxiliary trapping electrode in an external source Fourier transform ion cyclotron resonance mass spectrometer

Abstract A scheme for the enhanced accumulation of ions injected into the trapped ion cell of a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer from an external electrospray ionization source is described. This method utilizes an auxiliary electrode positioned outside the trapped ion cell which increases the effective collisional path length for ions traversing the trapped ion cell. A comparison made between pressure-assisted accumulated trapping in a symmetric trapping well, an asymmetric trapping well, and a symmetric trapping well with that for the addition of an auxiliary electrode indicates that trapping efficiency can be significantly improved when an auxiliary electrode trapping configuration is utilized.